CN101034090A - Hepatitis E virus antibody and method and kit for detecting hepatitis E virus using same - Google Patents
Hepatitis E virus antibody and method and kit for detecting hepatitis E virus using same Download PDFInfo
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Abstract
This invention relates to a monoclonal antibody aim at HEV ORF2 antigen, and the restrain rate less than 50 percent by using competition ELISA, optimizing less than 25 percent. This invention also relates to a antibody compound, which maintain at lest one sort monoclonal antibody above described; a kit which using double antibody sandwich ELISA method to detect HEV antigen, maintain antibody compound above described; and method through double antibody sandwich ELISA method and utilize antibody compound to detect HEV antigen in sample. This invention also relates to the using of described antibody compound in detecting HEV antigen.
Description
Invention field
The present invention relates to a kind of monoclonal antibody, and the inhibiting rate that uses competitive ELISA and described monoclonal antibody is less than 50% at HEV ORF2 antigen, preferred less than 25% monoclonal antibody.The invention still further relates to a kind of antibody compositions, wherein contain at least a aforementioned monoclonal antibody; A kind of kit that uses double-antibodies sandwich ELISA to detect HEV antigen, it contains aforementioned antibody compositions; And utilize aforementioned antibody compositions to pass through the in-vitro method of HEV antigen in the double-antibodies sandwich ELISA test sample.The invention still further relates to described antibody compositions and be used to detect the purposes of HEV antigen.
Background technology
Hepatitis E virus (HEV) is intestinal transmitted non-A non-B hepatitis (EntericallyTransmitted non-A, non-B hepatitis, main pathogens ET-NANBH).Think in the past, Hepatitis E mainly is popular in Africa and under-developed area such as Asia, along with in recent years to hepatitis E virus (HEV) research deeply and coherent detection means perfect, be seen in report in European, the increasing non-Introduced cases HE case of the U.S. and Japan, the popularity of HEV is serious more than the previous imagination, so the testing of HEV virus more and more obtains paying attention to.
Epidemiological study shows, from infect HEV to the time that clinical symptoms occurs about 15-60 days, 1-6 week course of disease duration.And in experimental infection, the volunteer infects behind the HEV and to occur clinical symptoms in about 36 days.
Existing Hepatitis E aetology detects and mainly contains following several method:
1. immunology detection
Immunological detection method mainly adopts enzyme-linked immune detection method at anti-HEV antibody, detects anti-HEV IgM, IgA and IgG antibody in the serum.
Because the different types of HEV antibody have different meanings for the HEV INFECTION IN DETECTION, so HEV antibody test reagent need detect respectively at the different type antibody of anti-HEV.Present HEV IgG detectable adopts indirect method, and this method specificity is not high, and anti-HEV IgG antibody duration after infection is very long, therefore, only detects IgG antibody and can not effectively distinguish previous infection, recent infection and acute infection.In addition, anti-HEV IgG antibody is 1-5% at non-popular regional positive rate, but HEV popular regional more than 25 years old among the crowd positive rate can reach 40%, there is certain interference effect in the diagnosis of Hepatitis E acute infection,
IgM antibody has the diagnostic value higher than IgG as the sign of acute infection and/or recent infection.There is the low shortcoming of affinity and specificity in IgM antibody as the antibody that occurs the earliest after infecting, and this specific character of IgM antibody has determined HEV IgM antibody test reagent to have all lower shortcoming of susceptibility and specificity.
And present IgM detectable adopts indirect method or prize law more, the prize law that Yu et al sets up highly sensitive in classical indirect method, but exist in the time of interior IgM-rheumatoid factor of human body and anti-HEV IgG and may cause false positive, because the IgM-rheumatoid factor can with the Fc section bridging of anti-HEV IgG, by sample being diluted the specificity that improves reagent, but reduced the susceptibility of reagent when doing so again.
Chau et al thinks, IgA antibody also can be used as HEV in the recent period or the sign antibody of acute infection, though the IgA antibody positive duration is still waiting clinical and further confirmation epidemiological study with meaning in the HEV Infect And Diagnose.
At present, the HEV antibody ELISA detectable of Gong Rening is the HEV antibody assay kit of Genelab company and Abott company in the world, and they all adopt HEV 1 type and 2 type antigens for detecting antigen.
2. molecular Biological Detection
Molecular Biological Detection is meant to be used the RT-PCR method to detect serum, ight soil and organizes HEV virus genome RNA in the equal samples.Yet, as intestinal transmitted virus, the HEV viremia virusemia duration is very short, spent the viremia virusemia phase during many hospitalizes or near latter stage, viral RNA copy number in the blood is very low, be difficult to detect by RT-PCR, this situation is especially outstanding in the underdeveloped developing country of medical and health condition.Though the ight soil toxin expelling duration slightly is longer than viremia virusemia, may there be the material that influences PCR in the sample collecting difficulty in vulnerable to pollution and the ight soil.There is very big difference in the positive rate that the RT-PCR of present report detects in patient HE, and from 30% to 80% does not wait, and this may mate with sample collecting time, sample retention situation, primer, the PCR condition is relevant with other uncertain factor.Therefore, the RT-PCR diagnostic method that detects HEV RNA at present only is used for laboratory study, does not also have commercial reagent by clinical employing.
Known HEV duplicates after infecting body, can cause the damage of hepatic tissue after the generation q.s virus, shows as hepatitis symptom clinically, and the laboratory is detected can show that then relevant enzymes is abnormal response, is apparent infection; Though yet virulent duplicating in some the infected's body do not cause typical clinical symptoms, is subclinical infection.No matter be dominance or negative the infection, in the early stage body that infects, all have HEV,, can detect nucleic acid and the antigen of HEV during this period if detection means is enough sensitive.Along with the progress of course of infection, virus can be brought out body and be produced immune response, and at first the inductive infection person produces anti-HEV IgM antibody, and certain time, IgG also can be along with generation after IgM produces, and continues the long time, also can produce IgA antibody simultaneously.Along with production of antibodies, the titre of body inner virus also can progressively reduce, last thoroughly removing.The dynamic change that detects IgM antibody and IgG antibody can be diagnosed the acute infection of HEV, but before antibody produced, the viral nucleic acid of HEV and antigen just existed, and therefore detects the purpose that HEV viral nucleic acid and antigen can reach more early diagnosis.
At present the HEV nucleic acid detection technique has been developed in some laboratory, but its trivial operations, technical requirement height, pollute easily, the more high drawbacks limit of cost its widespread use.If develop the lower antigen detection method of technical requirement, will provide very important instrument for the early diagnosis that HEV infects.
Infect in order promptly and accurately to detect HEV, press for more sensitive for a long time and method for quick accurately always.In order to address the above problem, the inventor is by adopting HEV ORF2 as the antigen immune mouse, screening is at the monoclonal antibody of described HEV antigen, having obtained can be with the kit of the micro-HEV antigen that exists in the direct test sample of high sensitivity, accurately detects in early days thereby realized infecting at HEV.
Summary of the invention
One aspect of the present invention relates to a kind of monoclonal antibody at HEV ORF2 antigen.
Another aspect of the present invention also relate to use competitive ELISA and monoclonal antibody of the present invention inhibiting rate less than 50%, preferred less than 25% monoclonal antibody.。
Another aspect of the present invention also relates to a kind of antibody compositions, wherein contains at least a aforementioned monoclonal antibody.
One side more of the present invention relates to a kind of kit that double-antibodies sandwich ELISA detects HEV antigen that is used for, and wherein contains aforementioned antibody compositions.
Another aspect of the present invention relates to and utilizes the in-vitro method of aforementioned antibody compositions by HEV antigen in the double-antibodies sandwich ELISA test sample.
The invention still further relates to described antibody compositions and be used to detect the purposes of HEV antigen.
Detailed Description Of The Invention
Existing confirmed HEV genome co-exists in 3 opening code-reading frames (ORF 1-3).The maximum albumen of ORF1 (about 28-5106nt) coding HEV virus, the about 186kDa of this albumen comprises at least 4 structural areas.Up to the present have only the ORF1 albumen that laboratory seldom can The expressed, the ORF1 albumen of partitioned representation is applied to function assessment research more, and the ORF1 fragment is only used in HEV antibody Western trace detectable at present, seldom uses in the ELISA detectable.
ORF2 gene length 1980bp (about 5147nt-7126nt), the coding molecule amount is about the viral capsid proteins of 72kDa.ORF2 albumen has one 111 amino acid whose burst and three potential glycosylation sites that may be used for secreting, expressing.Anti-ORF2 protein antibodies is significant in HEV infects, though the infection mechanism of HEV is not illustrated yet, but as the ORF2 albumen of HEV capsid protein in course of infection, playing the part of probably with host cell membrane on receptors bind and bring out virus uncoating and the role that enters cell, therefore the most neutralizing antibodies that have been found that at present all are anti-ORF2 protein antibodies.
The ORF2 albumen that is used for antibody test at present is mainly derived from the complete ORF2 albumen of insect cell system expression and the ORF2 protein fragments of Escherichia coli system expression, but different antigen shows different antibody binding capacities, can be more effective in conjunction with the anti-HEV antibody in convalescent's serum than ORF2 albumen total length such as the ORF2 protein fragments.Use in the synthetic antigenicity research process of peptide ORF2 albumen, find 12-147, the 381-504 of ORF2 albumen and 573-660 zone exist more can with the epitope of HE patients serum reaction, these zones are suitable for the structure of HEV antibody test reagent.
A little phosphorylated protein of ORF3 coding HEV virus, molecular weight is about 13.5kDa.It interacts with the liver cell skelemin that is called as AMBP.ORF3 albumen is less, its epitope that contains is also less relatively, 3 the ORF3 proteantigen epi-positions (31-40,63-76 and 105-122) that have been found that at present are linear epitope, wherein the C` of ORF3 albumen holds, 105-122aa particularly, antigenicity is higher, and the antibody positive rate at this epi-position among patient HE is higher, comprises the part fragment of ORF3 albumen in the present antibody diagnosing reagent more.
In order to obtain directly to detect the high-sensitivity detecting method of HEV antigen, the inventor uses HEV ORF2 immune mouse, conventional method prepares hybridoma, uses immune primordial covering elisa plate to detect and screen, and prepares 24 strain of hybridoma altogether and is used for further screening.
What unless otherwise defined, used here all technology and scientific term were all expressed is in the common implication that those of skill in the art understood that the present invention relates in the field.Here used name and be widely used conventional steps in the corresponding field in cellular incubation, molecular genetics, nucleic acid chemistry, Immunology Lab operation steps.
Antibody comprises the polypeptied chain aggregate that links together by disulfide bond.Two polypeptide main chains that are called light chain and heavy chain constitute all primary structure classifications (isoreagent) of antibody.Heavy chain and light chain all are further divided into some subprovinces that are called variable region and constant region.Heavy chain comprises single variable region and three different constant regions, and light chain then comprises single variable region (variable region that is different from heavy chain) and single constant region (constant region that is different from heavy chain).The binding specificity of antibody is responsible in the variable region of heavy chain and light chain.
" variable region of heavy chain " is meant a peptide species, and (1) its length is 110 to 125 amino acid; (2) the heavy chain amino acid sequence that begins from heavy chain N end amino acid corresponding to monoclonal antibody of the present invention of its amino acid sequence.Equally, " variable region of light chain " is meant a peptide species, and (1) its length is 95 to 115 amino acid; (2) the light chain amino acid order that begins from light chain N end amino acid corresponding to monoclonal antibody of the present invention of its amino acid sequence.
Used term Fab, Fc, F (ab)
2And Fv respectively has the immunology implication of its standard (to see Klein, immunology, John Wiley, New York, 1982; Parham, Chapter14, in Weir, ed. immunochemistry, 4
ThEd., Blackwell Scientific Publishers, Oxford, 1986).
An aspect of of the present present invention relates to a kind of monoclonal antibody at HEV ORF2 antigen.
In one embodiment of the invention, adopt known technology to set up hybridoma cell line of the present invention.This method generally includes a kind of clone that can go down to posterity lastingly and a kind of bone-marrow-derived lymphocyte that produces required antibody merges.Obtaining suitable lymphocytic technology from the mammal of having injected target antigen knows.In general, people's cell can use peripheral blood lymphocyte if desired; The non-human mammal cell then uses splenocyte or lymph-node cell if desired.Give the antigen of host mammal duplicate injection purifying, and make this mammal produce required antibody-producting cell, collect these cells then and merge with the clone that can go down to posterity lastingly.Cell-fusion techniques also is to know in this area.Generally comprise cell is mixed with fusion agent such as polyglycol.Select hybridoma with standard method such as HAT back-and-forth method.Use the immunoassay of standards such as ELISA method to analyze the nutrient culture media of these hybridomas to select the hybridoma of the required antibody of secretion.The purified technology of protein of use standard is by reclaiming antibody in the nutrient culture media.When using any above-mentioned technology, have many documents can be for reference (people such as Kohler, hybridoma technology (Hybridoma Techniques), Cold Spring Harbor Laboratory, NewYork, 1980; Tijssen, the practice of enzyme immune detection and theoretical (Practice andTheory of Enzyme Immunoassays), Elsevier, Amsterdam, 1985; Or the like).
The application of antibody fragment and generation are also known.As Fab fragment (Tijssen, the practice of enzyme immune detection is with theoretical, Elsevier, Amsterdam, 1985) and Fv fragment (people such as Hochman, biological chemistry (Biochemistry), 12:1130-1135,1973; People such as Sharon, biological chemistry, 15:1591-1594,1976; People such as Ehrlich, United States Patent (USP) 4470925).In addition, can make up bispecific antibody with these compounds of the present invention and composition by known technology, as (Quadromas) (Reading of the further fusion (promptly forming so-called quadruple hybridoma) by hybridoma, United States Patent (USP) 4474493) or the chemistry of half point connect (people such as Brennan again, science (Science), 229:81-83,1985).
Also available recombination method makes distinctive antibody of hybridoma of the present invention and antibody fragment.Utilize the method well known in the art variable region sequences of coded sequence, the especially heavy chain of acquisition code book invention monoclonal antibody and the variable region sequences of light chain from hybridoma of the present invention easily.Those skilled in the art can utilize these coded sequences to analyze for the important amino acid region of monoclonal antibody binding specificity easily in this area, as being present in determinant complementary region (CDR) CDR1, CDR2 and the CDR3 of antibody gene light chain and variable region of heavy chain, its amino acid sequence corresponding has constituted the specific antigen calmodulin binding domain CaM of antibody.Utilize these amino acid sequences, can make the polypeptide that has with the same or similar specific bond ability of monoclonal antibody, express the single-chain antibody (ScFv) that forms as antibody heavy chain variable region gene and chain variable region gene are linked in sequence with recombination method; Antibody heavy chain variable region gene and chain variable region gene or CDRs gene substitution to the relevant position of human antibodies, are given expression to the human monoclonal antibody that more approaches human antibodies and have former monoclonal antibody binding specificity; Or the like.
According to said method, the inventor utilizes hepatitis E virus ORF2 immune mouse, the preparation monoclonal antibody.According to budapest treaty, the hybridoma cell line that the inventor will secrete monoclonal antibody of the present invention on Dec 30th, 2005 is deposited in Chinese typical culture collection center (CCTCC, China, Wuhan, Wuhan University), be respectively: preserving number is the hybridoma cell line HEV-NICPBP58 that the hybridoma cell line HEV-NICPBP04 of CCTCC-C0200519, hybridoma cell line HEV-NICPBP07 that preserving number is CCTCC-C0200520 and preserving number are CCTCC-C0200521.
Each monoclonal antibody prepared in accordance with the present invention and in conjunction with active fragment, those skilled in the art can obtain to encode described monoclonal antibody or its nucleotide sequence, and the different variants that produce according to the degeneracy of genetic codon in conjunction with active fragment.According to content disclosed by the invention, those skilled in the art can comprise the recombinant expression carrier of above-mentioned nucleotide sequence with the several different methods preparation, and by gained recombinant expression carrier transformed host cells.The selection of host cell and to be converted into those skilled in the art known is as long as it can express monoclonal antibody of the present invention or it is in conjunction with active fragment.
Another aspect of the present invention also relate to use competitive ELISA and monoclonal antibody of the present invention inhibiting rate less than 50%, preferred less than 25% monoclonal antibody.。Described monoclonal antibody is the monoclonal antibody of discerning same or similar epi-position with the secreted monoclonal antibody of the above-mentioned hybridoma cell line of the present invention.
Those of ordinary skills know, and can pass through selected marker, and to antigen titration behind the mark, competitive ELISA is measured the identification of loci.In one embodiment of the invention, the inventor carries out selectivity HRP mark by improvement sodium periodate method antagonist.Antibody concentration behind the mark is diluted to 50 μ g/ml, uses the elisa plate of immune primordial covering to carry out titration, and the antibody to mark in the titration process carries out doubling dilution.According to the grouping and the labelled antibody situation of monoclonal antibody, the back antibody that divides into groups is done competitive ELISA detect, whether similar monoclonal antibody discerns same or analogous epitope.With the identical irrelevant monoclonal antibody of competition antibody concentration as negative control, the unmarked antibody of and labelled antibody homophyletic identical with the competition antibody concentration is as positive control.37 ℃ hatch 1 hour after, wash plate, TMB colour developing, 450nm reads at place A value, the inhibiting rate of antibody to labelled antibody competed in calculating.Formula is as follows:
Inhibiting rate=(A
Competition-A
Positive/ A
Negative-A
Positive) * 100%
As inhibiting rate 〉=75% is that two strain monoclonal antibody institute recognition sites are uncorrelated fully; 〉=50%＜75% is not exclusively relevant; 〉=25%＜50% for relevant;＜25% for relevant fully.
Another aspect of the present invention, also relate to a kind of antibody compositions, wherein containing at least a is the secreted monoclonal antibody of hybridoma cell line HEV-NICPBP58 that the hybridoma cell line HEV-NICPBP04 of CCTCC-C0200519, hybridoma cell line HEV-NICPBP07 that preserving number is CCTCC-C0200520 and preserving number are CCTCC-C0200521 by preserving number.In a specific embodiments of the present invention, described antibody compositions contains the secreted monoclonal antibody of hybridoma cell line HEV-NICPBP58 that the hybridoma cell line HEV-NICPBP04 of promising CCTCC-C0200519, hybridoma cell line HEV-NICPBP07 that preserving number is CCTCC-C0200520 and preserving number are CCTCC-C0200521 simultaneously, and the ratio of preferred described antibody is 2: 5: 2.
One side more of the present invention, relate to a kind of kit that double-antibodies sandwich ELISA detects HEV antigen that is used for, comprise the monoclonal antibody at least a of the present invention of diagnosing effective dose or its in the described kit in conjunction with active fragment, or the aforesaid antibody compositions of the present invention.Those skilled in the art know, and suitably, also should comprise appropriate carriers in this kit, buffering agent/liquid and be used to detect the reagent of the signal that produces such as commercially available second antibody, and operation instructions.
Those skilled in the art know, and for solid phase reaction detects, monoclonal antibody of the present invention can be fixed in solid phase carrier, also testing sample can be fixed on the solid phase carrier.Reaction is at room temperature carried out a period of time usually, needs to wash the step of the sample that does not combine with monoclonal antibody of the present invention in the testing process.
For liquid phase reactor, can directly add monoclonal antibody of the present invention to the testing sample in being in specific buffer system usually, take place under the interactional temperature being suitable for antigen-antibody then, for example under the room temperature, reaction a period of time.
The source of monoclonal antibody of the present invention is depended in the selection of related second antibody in the present composition.Those skilled in the art know, and how to select corresponding second antibody according to the source of monoclonal antibody.Second antibody of the present invention can be a labelled with radioisotope, includes but not limited to use be selected from: 32P, 125I, S, 2H etc.; Can be non-labelled with radioisotope also, include but not limited to use be selected from: marks such as horseradish peroxidase, alkaline phosphatase, biotin, Streptavidin.The detection agent that uses in the detection method of the present invention depends on the label of the second antibody that testing process is used.Those skilled in the art know how to select suitable detectable.
Another aspect of the present invention relates to and utilizes the in-vitro method of aforementioned antibody compositions by HEV antigen in the double-antibodies sandwich ELISA test sample.In one embodiment of the invention, described method comprises the steps:
1) the secreted monoclonal antibody of hybridoma cell line HEV-NICPBP58 that adopts the hybridoma cell line HEV-NICPBP04 contain CCTCC-C0200519 simultaneously, hybridoma cell line HEV-NICPBP07 that preserving number is CCTCC-C0200520 and preserving number to be CCTCC-C0200521, preferably, the ratio of described antibody is 2: 5: 2, antibody compositions, as the capture antibody coated elisa plate;
2) in the ELIAS strip that is coated with capture antibody, add use and contain the test serum that 5% cow's serum PBST10 doubly dilutes, hatch washing;
3) add goat-anti HEV and resist more, hatch washing;
4) add the anti-sheep IgG of biotin labeling rabbit antibody then, hatch, washing;
5) add HRP mark Avidin again, hatch, washing;
6) add the substrate colour developing.
The invention still further relates to described antibody compositions and be used to detect the purposes of HEV antigen.
Below in conjunction with embodiment the present invention further is illustrated.
The generation of embodiment 1 hybridoma cell line
Get female Balb/c mouse in 6～8 ages in week, the 5 μ g antigens (cumulative volume 50 μ l) of every mouse muscle injection of initial immunity Fu Shi Freund's complete adjuvant emulsification.Carry out the immunity of secondary base plinth after 15 days, method is the antigen freund 's incomplete adjuvant emulsification of getting same amount, intramuscular injection.After 30 days, tail vein booster shots 5 μ g do not add the antigen of adjuvant, and behind booster immunization 72 hours, kill mouse, collect blood, get spleen and prepare splenocyte suspension (being suspended from the RPMI1640 nutrient culture media), cell counting count board cell count.Get the SP2/0 murine myeloma cell of cultivation by 1/6 in the quantity of splenocyte, centrifugal after mixing, utilize polyglycol (PEG 1500) that splenocyte and murine myeloma cell SP2/0 are merged.With after isopyknic feeder cells mix, (200 μ l/ hole) is in 37 ℃ of cultivations of 5% CO2gas incubator (ESPEC BNA-311) in the 96 porocyte culture plates that are placed in cell suspension.After 3 days, ((hypoxanthn, H) (thymidine, T) 0.388mg add RPMI1640 (GIBCO company) nutrient culture media to 100mL to the 1.361mg+ thymidine to xanthine with the HT nutrient culture media.After dissolving under 45～50 ℃ of conditions, filtration sterilization.) partly keep and change liquid.After 7 days, by plate, utilize as following enzyme linked immunosorbent assay (ELISA) and detect gained hybridoma supernatant nutrient solution in the 96 porocyte culture plates with NE2 recombinant antigen bag.For the cell clone of ELISA test positive, utilize limiting dilution assay to carry out cloning.
The ELISA detection method
Antigen through the HPLC purifying, be dissolved in 0.05mol/L carbonic acid bag and be cushioned liquid (20.02gNa2CO3,2.52g NaHCO3 adds deionized water to 1 liter, pH is 9.5) in, concentration is about 0.3 μ g/ml, 2 hours (37 ℃) of absorption place 4 ℃ to spend the night again on each hole surface of 96 hole tygon microtiter plates.With PBS-tween cleansing solution (8.0g NaCl, 0.2g KH
2PO
4, 2.9g Na
2HPO
412H
2O, 0.2g KCl and 0.5ml polysorbas20 add deionized water to 1 liter, and pH is 7.4) the washing titer plate to be to remove unconjugated antigen protein.Use then in the confining liquid (1 * PBS solution, form the same) in 200 μ l/ holes and add 2% gelatin, 0.2% casein and 2% sucrose) 37 ℃ of sealings 2 hours.Get rid of clean, pat dry final vacuum sealing, 4 ℃ of preservations.
During detection, in every hole, add 100 μ l cell culture fluid supernatants; Every 96 hole microtiter plate is established a positive control, adds 100 μ l mouse polyvalent antibodies (dilution in 1: 100 is used); Other establishes a negative control, adds 100 μ lHT cell culture fluids.37 ℃ of temperature were bathed 30 minutes, wash 5 times with PBS-tween cleansing solution, pat dry the back and add peroxidase-conjugated sheep anti mouse immunoglobulin (Ig) (HRP-GAM Ig, DAKO company), 37 ℃ of temperature were bathed 30 minutes, take out the back and wash 5 times with PBS-tween cleansing solution, (substrate solution A composition is: colour developing liquid A composition is: 13.42g Na successively to add each 50 μ l of substrate solution A, B after patting dry
2HPO
412H
2O, 4.2g citric acid H
2O and 0.3g hydrogen peroxide are 700ml with adjusted volume in the deionized water; Colour developing liquid B composition is: 0.2g tetramethyl benzidine, 20ml dimethyl formamide are 700ml with adjusted volume in the deionized water), 37 ℃ were developed the color 10 minutes, add 50 μ l stop buffer (2M H2SO4) cessation reactions, and on microplate reader, detect the OD450 value in each hole, usually with the OD450 value be higher than negative control more than 2 times the person be considered as the positive.
The acquisition of monoclonal antibody ascites and purifying
Get the healthy Balb/c mouse in 10 ages in week, lumbar injection freund 's incomplete adjuvant, every 0.5ml.After 2～7 days, collect the hybridoma of cloning, the centrifugal supernatant that goes adds the nutrient culture media that does not contain serum, regulates cell density to 2 * 10
5～2 * 10
6Individual/ml, every injected in mice 0.5ml.Mouse web portion increases after 7～10 days, begins to collect ascites.Centrifugal 15 minutes of 3000rpm, the liquid of clarification part in the middle of drawing, the filtering with microporous membrane degerming of 0.45 μ m ,-20 ℃ of preservations after the packing.
The ascites of handling well is used PBS (the 81ml 0.2mol/LNa of 0.02mol/L, pH7.4
2HPO
4, 19ml 0.2mol/L NaH
2PO
4, add physiological saline to 100ml) and the two-fold dilution, dropwise slowly add ammonium sulfate (concentration reaches 50% saturation degree) under stirring, 4 ℃ are spent the night.4 ℃, centrifugal 15 minutes of 12000rpm abandons supernatant, precipitation is dissolved among the PBS of 2 times of former ascites volumes.Dropwise slowly add ammonium sulfate under stirring, make ammonium sulfate concentrations reach 33% saturation degree, 4 ℃ of standing over night.4 ℃, centrifugal 15 minutes of 12000rpm abandons supernatant, precipitation is dissolved among the PBS of 2 times of former ascites volumes.Dropwise slowly add ammonium sulfate under stirring, (concentration reaches 50% saturation degree), 4 ℃ are spent the night.4 ℃, centrifugal 15 minutes of 12000rpm abandons supernatant.Precipitation is dissolved among an amount of PBS, in packing into the dialysis band, puts into 50～100 times and contain 20mmol/L NaCl, desalination about 12 hours under 4 ℃ of stirrings in the 120mmol/L Tris-HCl damping fluid of pH7.8, during change dislysate more than 3 times.Take out back packing-20 ℃ preservation.
With said method, filtered out 24 strains and secreted the hybridoma cell strain of anti-HEV-ORF2 monoclonal antibody.
Embodiment 2 MONOCLONAL ANTIBODIES SPECIFIC FOR, purifying, purity are identified
Mouse peritoneal injecting fluid paraffin 0.5ml/, after the week, pretreatment of mice lumbar injection hybridoma 10
6/ only, inducing ascites, week back extraction contains the ascites of monoclonal antibody, collects ascites 3000g centrifugal 30 minutes, draws supernatant, and-70 ℃ are frozen standby.
The ascites of gathering adopts sad-saturated ammonium sulphate precipitation method monoclonal antibody purification, adopts determined by ultraviolet spectrophotometry monoclonal anti bulk concentration behind the purifying, and adopts SDS-PAGE electrophoretic analysis monoclonal antibody purification purity.
Embodiment 3 antigens and antibody sandwich and sealing
Use 96 hole polystyrene plates (Polystyrene microtiter plates, Maxisorp F96, Nunk Laboratories, Glostrup Denmark) wraps quilt, uses 0.05M NaHCO
3Damping fluid (pH9.6) with antibody dilution to suitable concn, antigen diluent is 2.5 μ g/ml, every hole adds 100 μ l, 37 ℃ 2 hours, with pH7.4 contain 0.1%Tween PBS (PBST) washing 3 times after, every hole adds the bag that contains 20% cow's serum and is cushioned liquid 200 μ l, 37 ℃ of sealings 2 hours, after the PBST washing 3 times, be stored in 4 ℃ standby.
Indirect ELISA
In the indirect ELISA, use contain 5% cow's serum PBST with antibody dilution to be checked to suitable concn, add and be coated with in the microwell plate of antigen, hatched 1 hour for 37 ℃ in 100 μ l/ holes, PBST washing 5 times.Every then hole adds 100 μ l and uses the HRP mark sheep anti-mouse igg antibody contain 5% cow's serum PBST1:5000 dilution, 37 ℃ hatch 30 minutes after, PBST washing five times adds tmb substrate, 37 ℃ of colour developings 15 minutes.Read each hole A value at wavelength 450nm place.
Elisa plate uses 2.5 μ g/ml immunity primordial covering, the two anti-sheep anti-mouse igg antibodies that adopt the HRP mark, use indirect method that the monoclonal antibody of purifying is carried out the affinity bigness scale, it is 10 μ g/ml that monoclonal antibody purification all is diluted to concentration, doubling dilution is to 0.3ng/ml downwards, parallel diplopore, every plate are established negative control two holes, use irrelevant mouse resource monoclonal antibody as negative control.After the TMB colour developing, 450nm reads the A value.Draw the broken line graph of A value and concentration under the same monoclonal antibody variable concentrations, and calculate four parametric lines, calculate A by four parametric lines
50The time monoclonal anti bulk concentration, with A
50Concentration is represented the affinity of this strain monoclonal antibody.The results are shown in Table 1
Table 1 monoclonal antibody is measured the affinity of antigen
| Numbering | A 50 | Numbering | A 50 | Numbering | A 50 | Numbering | A 50 |
| 2 3 4 7 8 15 | 3.2×10 5 1.7 66.1 16.8 71.6 29.8 | 17 20 24 26 27 29 | 1.9 25.0 34.3 386.2 67.5 555.4 | 34 40 44 50 52 56 | 0.72 1436.8 1752.5 22.2 12.1 32.3 | 58 67 72 80 90 97 | 1.6×10 -4 32.9 15.5 11.8 27.5 23.4 |
Embodiment 4 adopts competitive ELISA to measure the identification epi-position of described monoclonal antibody
Competitive ELISA:
According to the titration results of labelled antibody, labelled antibody concentration dilution to A value is about about 2.00, will compete antibody concentration dilution 10 times simultaneously for the existing concentration of labelled antibody.Each 50ul of competition antibody and labelled antibody adds in the microwell plate, and parallel diplopore is measured.With the identical irrelevant monoclonal antibody of competition antibody concentration as negative control, the unmarked antibody of and labelled antibody homophyletic identical with the competition antibody concentration is as positive control.Hatched 1 hour for 37 ℃, PBST washing 5 times adds the colour developing of tmb substrate damping fluid, and wavelength 450nm reads the A value, calculates the inhibiting rate of competition antibody to labelled antibody.
Inhibiting rate=(A
Competition-A
Positive/ A
Negative-A
Positive) * 100%
Improvement sodium periodate method is carried out selectivity HRP mark to monoclonal antibody of the present invention, respectively mark: 4,7,8,15,26,27,29,34,40,44,52,56,58,72,80 and No. 97 antibody.Antibody concentration behind the mark is diluted to 50 μ g/ml, uses the elisa plate of immune primordial covering to carry out titration, and the antibody to mark in the titration process carries out doubling dilution.According to the grouping and the labelled antibody situation of monoclonal antibody, the back antibody that divides into groups is done competitive ELISA detect similar monoclonal antibody and whether discern same or similar epitope.Calculate the inhibiting rate of competition antibody to labelled antibody:
Inhibiting rate=(A
Competition-A
Positive/ A
Negative-A
Positive) * 100%
As inhibiting rate 〉=75% is that two strain monoclonal antibody institute recognition sites are uncorrelated fully; 〉=50%＜75% is not exclusively relevant; 〉=25%＜50% for relevant;＜25% for relevant fully.Table 2: competitive ELISA result
| Pairing | Inhibiting rate (%) | Pairing | Inhibiting rate (%) | Pairing | Inhibiting rate (%) |
| 2-72 3-72 50-72 2-80 3-80 29-80 40-80 44-80 72-80 2-29 | 88 0 4 89 20 104 114 91 20 40 | 3-29 40-29 44-29 2-40 40-44 56-97 90-97 56-90 | 104 51 43 107 4 15 -4 -13 | 26-67 24-26 17-52 34-58 4-7 8-7 20-7 20-8 4-8 | 5 88 6 1 92 65 66 0 82 |
Annotate: the antibody in back is labelled antibody in the pairing
The situation that suppresses result and grouping according to competition:
What recognition site was relevant fully in the 23 strain monoclonal antibodies is:
3-50-72-80；40-44；56-90-97；26-67；17-52；34-58；8-20
What recognition site was relevant is:
2-29；40-44-29
What recognition site was not exclusively relevant is:
40-44-29；8-7；20-7
The monkey serum that embodiment 5 uses HEV1 type, 4 types and pig source HEV to attack poison 2 weeks of back is selected suitable coated antibody
LAB indirect sandwich assay ELISA
Be coated with to add in the ELIAS strip of monoclonal antibody and use the test serum that contains 10 times of dilutions of 5% cow's serum PBST, hatched 2 hours PBST washing 3 times for 37 ℃.How anti-add goat-anti HEV, hatched 1 hour for 37 ℃, and PBST washing 5 times adds the anti-sheep IgG of biotin labeling rabbit antibody, 37 ℃ hatch 30 minutes after, PBST washing 5 times adds HRP mark Avidin again, 37 ℃ hatch 30 minutes after, PBST washing 5 times adds tmb substrate, and 37 ℃ were developed the color 15 minutes.Read each hole A value at wavelength 450nm place.
In order to determine to be suitable for wrapping the monoclonal antibody of quilt, the monoclonal antibody bag that uses 10 μ g/ml respectively is by the EILSA plate, adopt the HEV1 type of LAB indirect sandwich assay ELISA to 20 times of dilutions, the monkey serum that 4 types and pig source HEV attack poison 2 weeks of back detects, and chooses 11 strain antibodies that can detect 3 kinds of different HEV simultaneously and further screens.
Table 3: different monoclonal antibodies during as coated antibody to 1,4 and pig type HEV attack the S/Co value that poison back monkey serum detects
| Numbering | 2 | 3 | 4 | 7 | 8 | 15 | 17 | 20 |
| HEV1 HEV4 HEVs | 0.9 0.4 0.7 | 1.1 5.1 5.0 | 9.0 10.1 9.8 | 8.9 13.4 13.7 | 1.8 12.9 10.2 | 13.6 14.6 13.9 | 15.3 15.4 15.4 | 5.9 9.0 8.4 |
| Numbering | 24 | 26 | 27 | 29 | 34 | 40 | 44 | 50 |
| HEV1 HEV4 HEVs | 0.7 0.6 0.6 | 1.5 4.7 5.9 | 0.6 0.3 0.5 | 1.0 0.4 0.6 | 8.1 9.3 8.9 | 1.0 0.5 0.7 | 1.0 0.5 0.7 | 5.1 8.8 7.8 |
| Numbering | 52 | 56 | 5 8 | 67 | 72 | 80 | 90 | 97 |
| HEV1 HEV4 HEVs | 1.0 14.9 13.0 | 0.9 1.0 1.1 | 1.6 3.7 2.9 | 4.0 4.7 4.7 | 1.0 2.2 1.6 | 0.8 0.5 0.6 | 1.0 2.4 2.4 | 1.1 1.4 1.2 |
Embodiment 6. screening of suitable coated antibody-monoclonal antibody
27 parts of anti-HEV IgG that use You An hospital collects and the two positive serum of IgM further screen the 11 strain monoclonal antibodies of selecting., determine that finally the monoclonal antibody that 5 strains are suitable for wrapping quilt is respectively 4,7,15,34, No. 58.The results are shown in Table 4
Table 4: the S/Co value that 27 parts of anti-HEVIgG that different monoclonal antibodies are collected You An hospital during as coated antibody and the two positive serum of IgM detect
The selection of embodiment 7. coated antibody compositions
The collocation of selecting suitable monoclonal antibody to carry out the Sheet clonal antibody is selected, and the blend proportion of different monoclonal antibodies is groped, and the bag of monoclonal antibody is groped by concentration, and 27 parts of clinical samples are redeterminated.
According to above monoclonal antibody the reactivity of 27 parts of serum 6 combinations have been determined, using total concentration is that the isoconcentration potpourri bag of above monoclonal antibody combination of 10 μ g/ml is by elisa plate, the LAB indirect sandwich assay detects above-mentioned 27 parts of clinical serum, according to testing result, it is the highest to determine that 58-4-7 makes up recall rate.The results are shown in Table 5.
27 parts of anti-HEV IgG and the IgM pair of S/Co value that positive serum detect that the combination of table 5 different antibodies is collected You An hospital
The selection of the best proportioning of embodiment 8. coated antibody compositions
Each monoclonal antibody ratio in the 58-4-7 combination is groped, total concentration is 10 μ g/ml, total concentration is divided into 9 parts, detects the 27 parts of anti-HEV IgG under the different blend proportion situations of 58-4-7 You An hospital being collected and the detection effect of 7 parts of representative serum in the two positive serum of IgM respectively.Final definite 58-4-7 blend proportion is 2: 5: 2.The results are shown in Table 6.
The S/Co value that under the different blend proportion situations 7 parts of representative serum is detected in the table 6.58-4-7 antibody compositions
| 7∶1∶1 | 6∶1∶2 | 6∶2∶1 | 5∶1∶3 | 5∶2∶2 | 5∶3∶1 | 4∶1∶4 |
| 4.8 1.5 5.1 0.5 0.8 1.9 0.4 | 4.5 1.2 4.2 0.6 1.0 2.0 0.4 | 5.1 1.7 5.0 0.7 1.2 2.9 0.5 | 6.2 1.6 5.0 0.5 1.1 1.9 0.4 | 6.4 1.8 6.2 0.7 1.3 2.2 0.4 | 6.4 2.2 6.4 0.8 1.3 3.1 0.4 | 7.5 2.6 6.6 0.8 1.3 2.5 0.5 |
| 4∶2∶3 | 4∶3∶2 | 4∶4∶1 | 3∶1∶5 | 3∶2∶4 | 3∶3∶3 | 3∶4∶2 |
| 6.7 2.4 6.4 0.7 1.4 2.3 0.5 | 7.4 2.5 7.2 0.8 1.4 3.0 0.5 | 7.8 2.5 7.3 0.8 1.5 2.7 0.5 | 6.5 2.1 6.7 0.6 1.3 3.0 0.6 | 7.2 2.8 8.4 0.9 1.8 3.0 0.6 | 7.4 3.1 8.3 1.0 1.8 3.9 0.5 | 10.7 3.7 10.8 1.0 2.0 4.4 0.7 |
| 3∶5∶1 | 2∶1∶6 | 2∶2∶5 | 2∶3∶4 | 2∶4∶3 | 2∶5∶2 | 2∶6∶1 |
| 7.8 2.9 8.2 0.9 1.5 4.0 0.6 | 9.6 3.0 8.3 0.8 1.9 4.0 0.7 | 9.1 3.0 8.0 0.8 1.5 3.3 0.6 | 8.6 3.3 9.5 0.7 1.9 4.6 0.7 | 10.0 4.1 11.4 1.0 2.1 5.4 0.7 | 10.9 5.0 12.9 1.1 2.6 5.8 0.8 | 8.1 3.0 7.3 0.9 1.5 3.9 0.6 |
| 1∶1∶7 | 1∶2∶6 | 1∶3∶5 | 1∶4∶4 | 1∶5∶3 | 1∶6∶2 | 1∶7∶1 |
| 9.9 3.8 9.2 0.8 1.8 3.7 0.8 | 8.7 3.9 8.8 0.9 1.9 3.1 0.8 | 9.7 4.0 9.1 0.9 1.4 3.3 0.8 | 10.1 3.5 9.4 0.8 1.6 3.0 0.8 | 9.0 3.0 8.0 0.5 1.0 3.8 0.7 | 9.4 3.3 11.6 1.0 2.2 3.9 0.7 | 11.6 3.9 11.3 1.0 1.4 3.7 1.0 |
Embodiment 9. the suitableeest bags are determined by concentration
According to monoclonal antibody 58-4-7 ratio is that 2: 5: 2 bags are by elisa plate, bag is respectively 20 μ g/ml, 10 μ g/ml, 5 μ g/ml, 2.5 μ g/ml and 1.25 μ g/ml by concentration, 30 parts of serum are redeterminated, according to positive recall rate and negative A value, determine that only bag is 5 μ g/ml by concentration.
Claims (10)
1. monoclonal antibody at HEV ORF2 antigen, it is:
1) HEV-NICPBP04 is the hybridoma secretion of CCTCC-C200519 by preserving number;
2) HEV-NICPBP07 is the hybridoma secretion of CCTCC-C200520 by preserving number; With
3) HEV-NICPBP58 is characterized in that by preserving number being the hybridoma secretion of CCTCC-C200521.
2. monoclonal antibody at HEV ORF2 antigen, the competitive ELISA inhibiting rate of monoclonal antibody that it is characterized in that described monoclonal antibody and claim 1 is less than 50%, preferably less than 25%.
3. an antibody compositions wherein contains the monoclonal antibody at least a claim 1 or 2.
4. the antibody compositions of claim 3, it is made up of the monoclonal antibody HEV-NICPBP04 described in the claim 1, HEV-NICPBP07 and HEV-NICPBP58.
5. the antibody compositions of claim 4, wherein the ratio of monoclonal antibody HEV-NICPBP04, HEV-NICPBP07 and HEV-NICPBP58 is 2: 5: 2.
6. one kind is used for the kit that double-antibodies sandwich ELISA detects HEV antigen, it is characterized in that containing the antibody compositions of claim 3.
7. in-vitro method that utilizes HEV antigen in the double-antibodies sandwich ELISA test sample.
8. the method for claim 7, wherein said antibody compositions is made up of monoclonal antibody HEV-NICPBP04, HEV-NICPBP07 and HEV-NICPBP58.
9. the method for claim 7, the ratio of monoclonal antibody HEV-NICPBP04, HEV-NICPBP07 and HEV-NICPBP58 is 2: 5: 2 in the wherein said antibody compositions.
10. the antibody compositions of claim 3 is used to detect the purposes of HEV antigen.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103235120A (en) * | 2013-04-22 | 2013-08-07 | 苏州华益美生物科技有限公司 | Kit for compound detection of hepatitis E virus antibody profile as well as application of kit |
| CN103823057A (en) * | 2014-03-07 | 2014-05-28 | 中国农业科学院兰州兽医研究所 | Colloidal gold test strip for quick diagnosis of total swine HEV (Hepatitis E Virus) antibody |
| CN103837680A (en) * | 2014-03-07 | 2014-06-04 | 中国农业科学院兰州兽医研究所 | Method for preparing porcine hepatitis E virus (HEV) total antibody enzyme-linked immuno sorbent assay (ELISA) detection kit |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103235120A (en) * | 2013-04-22 | 2013-08-07 | 苏州华益美生物科技有限公司 | Kit for compound detection of hepatitis E virus antibody profile as well as application of kit |
| CN103823057A (en) * | 2014-03-07 | 2014-05-28 | 中国农业科学院兰州兽医研究所 | Colloidal gold test strip for quick diagnosis of total swine HEV (Hepatitis E Virus) antibody |
| CN103837680A (en) * | 2014-03-07 | 2014-06-04 | 中国农业科学院兰州兽医研究所 | Method for preparing porcine hepatitis E virus (HEV) total antibody enzyme-linked immuno sorbent assay (ELISA) detection kit |
| CN103823057B (en) * | 2014-03-07 | 2016-01-20 | 中国农业科学院兰州兽医研究所 | Total antibody colloidal gold fast diagnose test paper bar of one boar HEV and preparation method thereof |
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